Insects and other pests cost farmers billions of dollars annually in crop losses and the expense of keeping these pests under control. The damage caused by insect pests result in decrease crop yield and reduction of crop quality, ultimately leading to an overall increase in agricultural farming costs. To combat insect pest, chemical pesticides are utilized to prevent or mitigate against crop destruction. However, use of chemical pesticides has its disadvantages. Disadvantages include indiscriminate insect eradication which results in extermination of non-target, beneficial insect species. Chemical pesticide usage also leads to chemical residue run-off into streams and seepage into water supplies resulting in environment damage. Crop or insects consumed having chemical residues present a danger to animals higher up on the food chain. The handling and application of chemical pesticides also presents a danger as there is a possibility of accidental exposure to people handling the chemical pesticides or accidental disbursal into an unintended environmental area. In addition, prolonged chemical pesticide application results in the targeted-surviving pest developing an evolutionary resistance to the chemical pesticide. In order to eradicate the resistant-pest, a cycle of even more potent chemical pesticides are utilized, resulting in more environmental damage and eventually an even more chemical-resistant pest. As such there is a need in the art to control pest populations without the disadvantages of chemical pesticides.
An approach to decrease agricultural dependence of chemical pesticides is gene targeting of functional nucleic acids of target-pests by either over expressing or silencing gene expression. One approach is to utilize RNA interference pathways to knockdown essentially any gene of interest via double strand RNA. Double strand RNA (dsRNA) induces sequence—specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). RNAi is a post-transcriptional, highly conserved process in eukaryotes that lead to specific gene silencing through degradation of the target mRNA. The silencing mechanism is mediated by dsRNA that is homologous in sequence to the gene of interest. The dsRNA is processed into small interfering RNA (siRNA) by an endogenous enzyme call DICER inside the target pest, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex (RISC), which finds and cleaves the target mRNA. The dsRNA inhibits expression of at least one gene within the target, wherein inhibition of the gene exerts a deleterious effect upon the target.
Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire discloses a method to inhibit expression of a target gene in a cell in vitro comprising introduction of a ribonucleic acid into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide strands are separate complementary strands that hybridize to each other to form said double-stranded molecule, and the double-stranded molecule inhibits expression of the target gene.
Since dsRNA has different effectiveness in gene knockdown, utilizing RNA interference technique to develop an effective pesticide poses a challenge. In some instances, robust knockdown occurs, while other instances results in no knockdown despite efficient transfection. In addition, when a target protein is very abundant in an organism or has a very long half-life (days to weeks), the obvious RNAi effect will be difficult to observe. Liu, et al. (U.S. Pat. No. 6,846,482) discloses an expression vector engineered to produce dsRNA within a pest. Salient to Liu is contacting an insect with a recombinant baculovirus wherein a first ribonucleic acid whose sequence corresponds to at least a portion of at least one gene endogenous to the insect to control the insect. Given the advances made in the field of transfection efficiency and RNA interference, there is a need in the art to utilize the advances without a baculovirus as a vector. Such a method would mediate control of a target-pest without depending on variables associated with a baculovirus, such as utilizing baculoviruses that are host specific.
To utilize RNA interference as a method to regulate a gene expression, a specific biological pathway needs to be targeted. One such explored pathway is apoptosis. Apoptosis is an evolutionarily conserved pathway of cell suicide that is critical for development and homeostasis of an organism. Apoptosis is the deliberate programmed cell death in multi-cellular organisms. Suicidal cell death is orderly biochemical programmed cell death where cellular signals determine whether there is a deliberate life relinquishment by the cell. The balance between positive signals (surviving signals) and negative signals (death signals) decides whether a cell should commit suicide. Regulation of programmed cell death pathways facilitates a method to utilize a nucleic acid pesticide for pest control by either developing nucleic acid pesticides for promoting the expression of pro-apoptotic factors (gene over-expression) and/or develop nucleic acid pesticide to reduce the expression of anti-apoptotic factors (gene silencing) so that the pest will die. By utilizing the targeted-pest cell death pathway, there will be less dependence on chemically based pesticides for pest control.
Apoptosis was first experimentally examined in the breakdown of the intersegmental muscles of silkworm, Bombyx mori (L.) Cells undergoing apoptosis have distinguishing characteristics, such as cell shrinkage, genomic DNA fragmentation, chromatin degradation and condensation, circular cellular character indication of caspases activation, pyknosis, or karyorrhexis. Researchers have identified molecular components of the cell death machinery that involve cysteine aspartic acid proteases, for example caspase-9, caspase-3, caspase-7, caspase-8, and/or caspase-10. Other molecular components include Fas receptors, Bcl-2 and Bax proteins. In addition, there are other proteins, such as inhibitor of apoptosis (IAP) and second mitochondria-derived activator of caspases (SMAC) that regulate the apoptotic process.
Programmed cell death can be triggered by internal signals through the intrinsic mitochondrial pathway. In a healthy cell, the outer membrane of the mitochondria displays the apoptosis suppressing protein Bcl-2 on its surface. Internal cellular perturbation causes Bcl-2 to activate a related protein, Bax to puncture the outer mitochondrial membrane causing the heme protein, cytochrome c, to be released by the mitochondria. The release of cytochrome c binds to the cytosolic protein apoptotic protease activating factor-1 (Apaf-1). Using energy provided by ATP, these complexes aggregate to form apoptosomes. Apoptosomes then bind to and activate the cysteine protease caspase-9. Caspase-9 is one of a family of over a dozen caspases. Caspase-9 cleaves and activates caspase-3 and caspase-7. The activation of these executioner caspases creates an expanding cascade of proteolytic activity which leads to digestion of structural proteins in the cytoplasm and degradation of chromosomal DNA, and phagocytosis of the cell. The art discloses various proteins involved in the apopotosis pathway. For instance, Nunez et al. (U.S. Pat. No. 6,348,573) discloses a protein sequence for RIP-like interacting CLARP kinase that functions as a positive regulator of apoptosis.
Programmed cell death can also be triggered by external signals through the extrinsic or death receptor pathway. Signal molecules such as Apo-1 or CD95, (Fas) and Tumor necrosis factor (TNF) receptor are integral membrane proteins with receptor domains exposed at the surface of the cell. Binding of their respective complementary death activators FasL and TNF transmits a signal to the cytoplasm that leads to activation of caspase 8. Caspase 8 initiates a cascade of effector caspase activation and the positive feedback of biological perturbation eventually leads to phagocytosis of the cell.
Programmed cell death can also be triggered by apoptosis-Inducing Factor (AIF). AIF is a flavoprotein that is a mitochondria factor released during apoptosis that is normally located in the intermembrane space of mitochondria. Unlike intrinsic or extrinsic mitochondria pathways, cysteine-aspartic-acid-proteases are not utilized to initiate apoptosis with AIF. When the cell receives a sign for apoptosis, AIF is released from the mitochondria and translocates into the nucleus to bind to DNA. Experiments have shown that caspases are not involved in AIF-activated apoptosis pathway. DNA binding by AIF stimulates chromatin condensation and DNA fragmentation. DNA binding by AIF occurs through a distinct domain of the protein in a manner that does not rely on specific DNA sequences. AIF also has another domain that acts as an NADH oxidase, a redox enzyme. The NADH oxidase activity of AIF is separable from its DNA-binding activity and is not required for AIF to induce apoptosis. Given the knowledge of apoptosis pathways, there is a need to utilize the knowledge to target specific pathways for pest control.
Another family of apoptosis regulatory proteins are inhibitor of apoptosis proteins (IAPs). IAP suppresses apoptosis by preventing the activation of procaspases and endogenously inhibits the enzymatic activity of mature caspases. IAPs were originally discovered in insect baculoviruses (Cydia pomonella granulosis virus and Orgyia pseudotsugata nuclear polyhedrosis virus) (Birnbaum, M J. et al., 1994. Journal of Virology, 68:2521-2528; Clem R J. et al., 1994. Mol. Cell Biol, 14:5212-5222; Crook N E. et al., 1993. Journal of Virology, 67:2168-2174). Since their first reports in baculoviruses, IAPs have been identified in many other organisms, such as mosquito iridescent viruses (Delhon G. et al., 2006. Journal of Virology, 80:8439:8449), insects (Hay B A. et al., 1995. Cell, 83:1253-1262; Muro I. et al., 2002. J. Biol Chem, 277:49644-49650), yeast (Walter D. et al., 2006. J. Cell Sci., 1843:1851), and humans (Ambrosini G. et al., 1997. Nat. Med., 3:917:921; Liston P. et al., 1996. Nature, 379:349:353; Vitte-Mony I. et al., 1997. J. Biol. Chem., 273:33915-33921). Many IAPs are capable of blocking apoptosis when overexpressed in cells in a plurality of species (Beidler D R. et al., 1995. J. Biol. Chem., 270:16526-16528, Hawkins C J., et al., 1996. Cell Death Differ., 5:569-576). The exact molecular mechanisms by which IAPs inhibit apoptosis are still under investigation, however, it has been shown that IAPs are able to inhibit the activity of cysteine proteases that cleaves different target proteins. IAPs are capable of blocking apoptosis through direct inhibition of proapoptotic proteins, such as Reaper, HID, and GRIM in Drosophila (Vucic D. et al., 1998. J. Biol. Chem., 273:33915-33921; Vucic D. et al., 1998. Mol. Cell Biol., 3300-3309b, Vucic D. et al., 1997. Proc. Natl. Acad. Sci. USA, 94:10183-10188).
In Drosophila, four IAPs (DIAP1, DIAP2, deterin, and bruce) have been reported (Hay B A. et al., 1995. Cell, 83:1253:1262; Jones G. et al., 2000. J. Biol. Chem., 275:22157-22165; Vernooy S Y. et al., 2002. Curr. Biol., 12:1164-1168). Four other insect IAP1s have also been identified, namely TnIAP1 from the cabbage looper Trichoplusia ni (Seshagiri S. et al., 1999. J. Biol. Chem., 274:36769-36773), SfIAP1 from the fall armyworm Spodoptera frugiperda (Huang Q. et al., 2000. Proc. Natl. Acad. Sci. USA, 97:1427:1432), BmIAP from the silkworm Bombyx mori (Huang Q. et al., 2001. Biochim. Biophys. Acta., 1499:191-198), and AtIAP1 from the mosquito Aedes triseriatus (Blitvich B J. et al., 2002. Insect Mol. Biol. 11:431:442).
Baculovirus IAP genes include sequences encoding a ring zinc finger-like motif, which is presumed to be directly involved in DNA binding, and two N-terminal domains that consist of a 70 amino acid repeat motif termed a BIR domain (Baculovirus IAP Repeat). The inhibitor of apoptosis proteins typically have one to three baculovirus IAP repeat (BIR) domains. The BIR domain is essential for anti-apoptotic activity (Takahashi R. et al., 1998. J. Biol. Chem., 273:7787:7790; Walter D. et al., 2006. J. Cell Sci., 119:1843-1851). Approximately 70 amino acids in length containing a conserved stretch of cysteines and histidines, BIR domain is well-known for its ability to bind and inhibit caspases (Takahashi et al., supra) and mediate protein-protein interaction (Hozak R R. et al., 2000. Mol. Cell. Biol., 20:1877-1885). As such, efforts to utilize mammalian IAPs have been disclosed. For example, Korneluk et al. (U.S. Pat. No. 6,156,535) discloses a substantially pure DNA encoding mammalian IAP polypeptides and methods of using such DNA to express the IAP polypeptides in cells and animals to inhibit apoptosis. Additionally, Rothe et al. (U.S. Pat. No. 6,821,736) discloses human cellular IAPs having a series of defined structural domain repeats and/or a RING finger domain.
Furthermore, Shi et al. (U.S. Pat. No. 6,992,063) discloses peptides and peptidomimetics capable of modulating apoptosis through their interaction with cellular IAPs (inhibitor of apoptosis proteins). The peptides and mimetics are based on the N-terminal tetrapeptide of IAP-binding proteins, such as Smac/DIABLO, Hid, Grim and Reaper, which interact with a specific surface groove of IAP. Shi discloses a compound that binds a BIR-3 domain of IAP and relieves IAP mediated inhibition of caspase activity.
Regulating programmed cell death pathways to develop nucleic acid pesticides has an advantage over conventional chemical-based pesticides inasmuch as nucleic acid pesticides developed through regulating the programmed cell death pathways are specific in terms of pest target. Unlike traditional chemical pesticides, a nucleic acid pesticide is designed to produce specific gene product for one species, thus not affecting non-target organisms. In addition, the utilization of nucleic acid pesticide will maximize safety and minimize environmental impact. As such, there is a need in the art for a method utilizing a nucleic acid pesticide that operates via the apoptosis pathway.
Furthermore, a novel control method will be very effective against resistant species since the engineered nucleic acid pesticides would be specific against resistant pest species. A need exists in the art for a method of producing a pesticide utilizing nucleic acid as a pesticide. In addition, there is a need for a method for producing a pesticide by regulating programmed cell death pathways to develop nucleic acid pesticides for pest control.